Protein kinases play a critical role in the control of cell growth and differentiation. Aberrant expression or mutations in protein kinases have been shown to lead to uncontrolled cell proliferation, such as malignant tumour growth, and various defects in developmental processes, including cell migration and invasion, and angiogenesis. Protein kinases are therefore critical to the control, regulation, and modulation of cell proliferation in diseases and disorders associated with abnormal cell proliferation. Protein kinases have also been implicated as targets in central nervous system disorders such as Alzheimer's disease, inflammatory disorders such as psoriasis, bone diseases such as osteoporosis, atheroscleroses, restenosis, thrombosis, metabolic disorders such as diabetes, and infectious diseases such as viral and fungal infections.
One of the most commonly studied pathways involving kinase regulation is cellular signaling from receptors at the cell surface to the nucleus. Generally, the function of each receptor is determined by its pattern of expression, ligand availability, and the array of downstream signal transduction pathways that are activated by a particular receptor. One example of this pathway includes a cascade of kinases in which members of the Growth Factor receptor Tyrosine Kinases deliver signals via phosphorylation to other kinases such as Src Tyrosine kinase, and the Raf, Mek and Erk serine/threonine kinase families. Each of these kinases is represented by several family members which play related, but functionally distinct roles. The loss of regulation of the growth factor signaling pathway is a frequent occurrence in cancer as well as other disease states. Fearon, Genetic Lesions in Human Cancer, Molecular Oncology, 1996, 143-178.
One receptor tyrosine kinase signaling pathway includes the vascular endothelial growth factor (VEGF) receptor kinase. It has been shown that binding of VEGF to the receptor VEGFR2 affects cell proliferation. For instance, binding of VEGF to the VEGFR-2/flt-1 receptor, which is expressed primarily on endothelial cells, results in receptor dimerization and initiation of a complex cascade which results in growth of new blood vessels (Korpelainen and Alitalo, Curr. Opin. Cell. Biol. 1998, 10, 159). Suppression of formation of new blood vessels by inhibition of the VEGFR tyrosine kinases would have utility in a variety of diseases, including treatment of solid tumors, diabetic retinopathy and other intraocular neovascular syndromes, macular degeneration, rheumatoid arthritis, psoriasis, and endometriosis.
The signals mediated by kinases have also been shown to control cell growth, cell death and differentiation in the cell by regulating the processes of the cell cycle. Progression through the eukaryotic cell cycle is controlled by a family of kinases called cyclin dependent kinases (CDKs). The loss of control of CDK regulation is a frequent event in hyperproliferative diseases and cancer.
Inhibitors of kinases involved in mediating or maintaining particular disease states represent novel therapies for these disorders. Examples of such kinases include inhibition of Src, raf, and the cyclin-dependent kinases (CDK) 1, 2, and 4 in cancer, CDK2 or PDGF-R kinase in restenosis, CDK5 and GSK3 kinases in Alzheimers, c-Src kinase in osteoporosis, GSK-3 kinase in type-2 diabetes, p38 kinase in inflammation, VEGF-R 1-3 and TIE-1 and -2 kinases in angiogenesis, UL97 kinase in viral infections, CSF-1R kinase in bone and hematopoietic diseases, and Lck kinase in autoimmune diseases and transplant rejection.
An additional kinase signal transduction is the stress-activated protein kinase (SAPK) pathway (Ip and Davis Curr. Opin. Cell Biol. 1998, 10, 205). In response to stimuli such as cytokines, osmotic shock, heat shock, or other environmental stress, the pathway is activated and dual phosphorylation of Thr and Tyr residues within a Thr-Pro-Tyr motif of the c-jun N-terminal kinases (JNKs) is observed. Phosphorylation activates the JNKs for subsequent phosphorylation and activation of various transcription factors, including c-Jun, ATF2 and ELK-1.
The JNKs are mitogen-activated protein kinases (MAPKs) that are encoded by three distinct genes, jnk1, jnk2 and jnk3, which can be alternatively spliced to yield a variety of different JNK isoforms (Gupta et al., EMBO J 1996, 15, 2760). The isoforms differ in their ability to interact with and phosphorylate their target substrates. Activation of JNK is performed by two MAPK kinases (MAPKK), MKK4 and MKK7. MKK4 is an activator of JNK as well as an additional MAPK, p38, while MKK7 is a selective activator of JNK. A number of MAPKK kinases are responsible for activation of MKK4 and MKK7, including the MEKK family and the mixed lineage kinase, or MLK family. The MLK family is comprised of six members, including MLK1, MLK2, MLK3, MLK6, dual leucine zipper kinase (DLK) and leucine zipper-bearing kinase (LZK). MLK2 is also known as MST Katoh, et al. Oncogene, 1994, 10, 1447). Multiple kinases are proposed to be upstream of the MAPKKKs, including but not restricted to germinal center kinase (GCK), hematopoietic progenitor kinase (HPK), and Rac/cdc42. Specificity within the pathway is contributed, at least in part, by scaffolding proteins that bind selected members of the cascade. For example the JNK interacting protein-1 (JIP-1) binds HPK1, DLK or MLK3, MKK7 and JNK, resulting in a module which enhances JNK activation (Dickens et al. Science 1997, 277, 693).
Manipulation of the activity of the SAPK pathway can have a wide range of effects, including promotion of both cell death and cell survival in response to various pro-apoptotic stimuli. For example, down-regulation of the pathway by genetic disruption of the gene encoding JNK3 in the mouse provided protection against kainic acid-induced seizures and prevented apoptosis of hippocampal neurons (Yang et al. Nature 1997, 389, 865). Similarly, inhibitors of the JNK pathway such as JIP-1 inhibit apoptosis (Dickens, supra). In contrast, the activity of the JNK pathway appears to be protective in some instances. Thymocytes in which MKK4 has been deleted display increased sensitivity to CD95- and CD3 mediated apoptosis (Nishina et al. Nature 1997, 385, 350). Overexpression of MLK3 leads to transformation of NIH 3T3 fibroblasts (Hartkamp et al. Cancer Res. 1999, 59, 2195).
An area the present invention is directed toward is identification of compounds that modulate the MLK members of the SAPK pathway and promote either cell death or cell survival. Inhibitors of MLK family members would be anticipated to lead to cell survival and demonstrate therapeutic activity in a variety of diseases, including chronic neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease and Huntington's disease and acute neurological conditions such as cerebral ischemia, traumatic brain injury and spinal injury. Inhibitors of MLK members leading to inhibition of the SAPK pathway (JNK activity) would also display activity in inflammatory diseases and cancer.
Thus, there is a need for novel classes of compounds which demonstrate activity toward receptor and non-receptor types of protein kinases. It has been discovered that a class of compounds, referred to herein as fused [d]pyridazin-7-ones, are useful as agents for the regulation of protein kinases. The present invention is therefore directed to, inter alia, their use as therapeutic agents for the treatment of the foregoing disorders.